Project Summary Bitter taste receptors (TAS2Rs) are expressed on human airway smooth muscle (HASM) and when activated markedly relax the muscle and dilate the airway. Utilization of this pathway, which is distinct from that of ?- agonists acting at ?2-adrenergic receptors (?2ARs), will provide a new class of direct bronchodilators for treating or preventing bronchospasm in asthma. The TAS2R14 subtype is highly expressed in HASM and is a prime target for developing a novel therapeutic agent. However, there are gaps in our knowledge about the molecular/cellular biology and physiology of HASM TAS2Rs, including how they couple to relaxation, the potential for tachyphylaxis due to short-term (receptor phosphorylation) and long-term (downregulation of receptor expression) events, and the potential to bias receptor signaling towards favorable signaling for asthma treatment. The broad, long-term objective of the Project is to improve our understanding of HASM TAS2R biology relevant to treating airway contraction in asthma. To fill these gaps in our knowledge, in Aim 1 we will define the mechanism by which TAS2Rs evoke relaxation, which we hypothesize is via inhibiting phosphorylation of the actin severing protein cofilin. Studies will be performed in cultured HASM cells derived from nonasthmatic as well as asthmatic donor lungs, the latter being important because of the potential for the disease to modify receptor function. Studies will include siRNA-based knockouts of cofilin, and the upstream components of the proposed pathway that link the receptor:G-protein:effector complex to cofilin. In Aim 2, agonist-prompted phosphorylation of TAS2R14 by GRKs will be studied using whole cell phosphorylation and receptor purification experiments. To define the precise residues phosphorylated by GRKs, TAS2R14 will be mutated to substitute potential Ser/Thr phospho-acceptor sites with Ala, thus defining a bar-code for ?arrestin binding. The consequences of phosphorylation on ?arrestin conformation and intracellular receptor signaling, and HASM relaxation, will then be determined. In Aim 3, a panel of TAS2R14 agonists will be utilized to determine the mechanisms by which a TAS2R agonist can be biased away from deleterious outcomes and towards advantageous outcomes in regards to asthma therapy. This endeavor will provide the basis for agonist-based ?tuning? of the receptor to be highly efficacious in bronchodilating and inhibiting HASM proliferation, but display little short- or long-term agonist-promoted desensitization or downregulation, such that clinical tachyphylaxis is not apparent. All three aims will utilize parallel physiological measurements of contraction and relaxation using nonasthmatic and asthmatic HASM cells, and an inflammatory precision-cut human lung slice model, in order to link biochemical events to clinically relevant physiological responses. Collectively, these studies will provide the basis for development of a novel class of direct bronchodilators which can be utilized alone, or in combination, with ?-agonists for the treatment of asthma.